1. Field of the Invention PA1 2. Discussion of Prior Art
The present invention relates to a solid phase system for ligand assay. More particularly, the invention relates to an immunological method for analyzing biological fluids wherein analytical reactions are conducted in an inert porous medium.
Variations of natural immunological reactions have been found very useful as analytical techniques. These reactions have been most useful in clinical laboratory procedures, but their use is not limited to clinical applications. Because of the specificity of the reactions, they are of particular advantage in analyzing complex biological fluids. Often, conventional chemical analyses are not capable of differentiating complex molecules in a biological fluid. Such fluids can be analyzed for a variety of components such as drugs, enzymes, hormones, etc. by contacting the fluid with an appropriate antibody. Likewise, analyses for specific antibodies can be conducted by contacting the fluid with an appropriate antigen. Other natural binding proteins are also quite useful in some types of assay procedures.
Unfortunately, the antibody-antigen reaction is generally not directly measurable, therefore, techniques have been devised for its indirect measurement. For instance, an antibody, antigen, or binding protein (collectively referred to as a ligand) may be labeled by various means. The amount of bound labeled ligand can thus be correlated to the concentration of the analyte in the biological fluid. Conventional labels include radioactive tags, e.g., .sup.125 I or tritium, enzymes, chromophores, fluorophores and enzymes cofactors and effectors. In the case of radioactive tags, the concentration of the labeled ligand is usually determined by placing the compound in a scintillation counter. Enzymes may be measured by reacting the labeled ligand with a substrate, which by the action of the enzyme, releases a chromogenic or fluorogenic substance that can be measured by conventional techniques. Ligands labeled with enzyme cofactors or effectors can be detected similarly by their effect on enzyme action on a substrate. Compounds labeled with chromophores may be directly measurable, e.g., by fluorescence, ultraviolet spectroscopy or other spectroscopic means.
Immunochemical assays generally fall into one of two classifications. In the competitive assay, a limited quantity of binding material is contacted with a solution containing the analyte and a known concentration of a labeled analyte. The labeled and unlabeled analyte compounds compete for the binding sites on the binding material. By reference to a calibration curve, the amount of labeled analyte bound to the binding material can be correlated with the concentration of the analyte in the test solution. A second type of immunological assay, the sandwich assay, involves contacting a binding material with a solution containing the analyte to cause the analyte to bind to the binding material. This complex is then contacted with a solution of a labeled binding material, generally an antibody, which reacts with the bound analyte. The amount of bound labeled binding material is thus directly proportional to the amount of bound analyte.
In all of the described methods, an essential step is to separate the unbound labeled material from the bound labeled material. A technique widely employed for such separation is to immobilize one of the reactants. For instance, an antibody may be adsorbed onto a solid support such as a test tube wall. After labeled material and analyte become bound to the immobilized antibody, the solid support is rinsed free of unbound labeled material. A variety of solid supports have been proposed for this purpose. Such supports include test tube walls, plastic cups, beads, plastic balls and cylinders, paper, and glass fibers.
In U.S. Pat. No. 3,888,629, June 10, 1975, K. D. Bagshawe discloses a reaction cell and an immunoanalytical method, in which antibody is immobilized in a matrix pad of absorbent material such as a glass fiber pad. The reference discloses an antibody impregnated sheet of the absorbent material. Disks or pads of the material are then punched from the mat and placed in a reaction cell. Solutions of the analyte and other reactants are placed on the pad where the immunological reaction occurs. A buffer is then filtered through the pad to wash out unreacted labeled material. Because of surface tension and capillary action, liquid does not easily pass through the pad; therefore, an absorbent material is placed under the pad to facilitate filtration.
To quantitate the reaction conducted in the Bagshawe cell, the pad must be removed from the cell and either placed in a gamma counter (in the case of a radioimmunoassay) or placed in some type of indicator solution (in the case of an enzymeimmunoassay).
A need exists for a rapid, quantitative solid phase ligand assay, which can be conducted entirely on a solid matrix. Advantageously, the method would not require special reaction cells, and the separation of unreacted labeled material could be effected cleanly without filtering large amounts of solvents through the matrix. The amount of binding material deposited in the reaction zone on the solid matrix should be accurately controllable. Such accurate control has not heretofore been realized in prior art methods.